1. Field of the Invention
The present invention relates to the field of pyridoxal-5'-phosphate (PLP) activated assays for transaminases such as glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT).
2. Brief Description of the Prior Art
Transamination is the transfer of an amino group from one molecule to another without the intermediate formation of ammonia. Enzymes that catalyze transaminations are referred to as aminotransferases or, more commonly, transaminases. The two substrate-specific transaminases which have been described in serum are glutamate oxalacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT). The mentioned transaminases catalyze the following reactions:
(a) GOT ##STR1## (b) GPT ##STR2## PA0 (a) GOT ##STR3## (b) GPT ##STR4## The method for GOT was introduced by LaDue et al in Science, Serum Oxalacetic Transaminase Activity in Human Acute Transmural Myocardial Infarction, 120:497 (1954). The method for GPT was described by Henley et al in J. Lab. Clin. Med., A New Method for the Determination of Glutamic Oxalacetate and Glutamic Pyruvic Transaminase in Plasma, 46:785 (1955). Alternatively, the glutamate from reaction (1) or (2) can be reacted with NAD in the presence of glutamate dehydrogenase to produce NADH, which can be measured. With various modifications and improvements, these are still the methods of choice for the measurement of serum transaminases. See, for example, U.S. Pat. No. 4,241,179.
The most important uses for serum measurements of GOT and GPT are in myocardial infarction and hepatobiliary disease. The methods normally used in the clinical laboratory to measure GOT and GPT involve kinetic, NADH-coupled (NADH is reduced nicotine adenine dinucleotide) reactions monitored at 340 nanometers (nm). These coupled reactions use malate dehydrogenase (MDH) and lactate dehydrogenase (LDH), respectively, and are as follows:
The reactions in both of these methods can be followed by a variety of techniques. See, for example, Henry et al (Eds.), Clinical Chemistry Principles and Technics, 2d Ed., Harper & Row, Publishers, pp. 873-892 (1974). NAD(H) can be measured either directly or after conversion of a redox indicator.
Alternatively, Deneke et al, U.S. Pat. No. 4,271,265, discloses a transaminase assay method in which the .alpha.-ketoglutarate formed, as in reactions (1) or (2) above, is reacted with .gamma.-aminobutyrate in the presence of .gamma.-aminobutyrate transaminase with formation of succinate semialdehyde. Nicotine adenine dinucleotide phosphate (NADP) is reduced with the latter in the presence of succinate semialdehyde dehydrogenase to give NADPH (NADPH is reduced nicotine adenine dinucleotide phosphate), and the latter is measured either directly or after conversion with a tetrazolium salt and an electron carrier such as diaphorase, phenantroline methosulphate or phenazine methosulphate to a formazan dye.
All transaminases appear to share a common reaction mechanism and appear to have the same coenzyme or prosthetic group. This coenzyme is pyridoxal-5'-phosphate (PLP): ##STR5## In transaminations PLP serves as the carrier for the .alpha.-amino group to be transferred. The aldehyde group of the PLP-apoenzyme complex accepts an amino group from the donor amino acid, e.g. aspartate or alanine, forming a Schiff base. By oscillating between the aldehyde and amino forms, the PLP transfers the amino group from an amino acid to a keto acid. Serum transaminases require PLP for full catalytic activity, and a portion of such transaminases are present in serum in the apoenzyme form. Addition of PLP to compositions for the determination of such transaminases provides more complete activation and, thus, greater sensitivity. This was suggested in Hamfelt, The Effect of Pyridoxal Phosphate on the Aminotransferase Assay in Blood, Scand. J. Clin. Lab. Invest. 18 (Suppl. 99):181 (1966). The addition of PLP to transaminase assay reagents has been observed to cause a "positive drift" or incorrectly high reading in the 340 nanometer range. It has been suggested that incorporation of anions at high concentrations eliminates this shift. Sanderson J., Clin. Chem. 27:1035, Abs. 054 (1981).